The present disclosure relates generally to devices, such as microfluidic chips, which can provide high-throughput detection of target analytes in samples, especially biological samples, using various assays. The disclosure also relates to processes for making and using such devices.
Enzyme-Linked Immunosorbent Assay (“ELISA”) is one of the most commonly used methods of various immunoassays. It has been widely used for detection and quantification of biological agents (mainly proteins and polypeptides) in the biotechnology industry, and is becoming increasingly important in clinical, food safety, and environmental applications. ELISA typically uses an enzymatic reaction to convert substrates into products having a detectable signal (e.g., fluorescence). Each enzyme in the conjugate can covert hundreds of substrates into products, thereby amplifying the detectable signal and enhancing the sensitivity of the assay.
In this regard, ELISA is a form of quantitative immunoassay based on the use of antibodies (or antigens) that are linked to an insoluble carrier surface. These antibodies (or antigens) are then used to capture the element antigen (or antibody) in the test solution. The antigen/antibody complex is then detected by measuring the activity of an appropriate enzyme that had previously been covalently attached to the antigen (or antibody).
The general principles and procedures used in typical ELISA are described here with reference to a 96-well microtiter plate:
(a) The first antibody (specific for the antigen to be assayed) is added to an ELISA plate. The first antibody is allowed to adsorb to the solid substrate surface. The excess antibody is removed from the plate after incubation.
(b) The wells are filled with blocking solution. The blocking solution provides proteins, which adsorb to all protein-binding sites and prevent subsequent nonspecific binding of antibody to the plate.
(c) The sample is added. If the sample contains the targeted antigen, it will bond to the adsorbed first antibody to form an antigen-antibody complex. After incubation, the plate is washed.
(d) The conjugate solution is added. The conjugate (the second antibody) is an appropriate enzyme-labeled ligand (usually an antibody), which will bond to the antigen. The conjugate solution is discarded and the plate is washed after incubation.
(e) The developing solution containing the substrate is added, which reacts with the enzyme in the conjugate. Each enzyme is able to convert hundreds of substrate into products to enhance the sensitivity of the assay. The products of the reaction emit fluorescence or change the color of the solution.
This process requires a series of mixing (reaction) and washing steps, which involves a tedious and laborious protocol. It often takes many hours to two days to perform one assay due to the long incubation times during each step. These long incubation times are mostly attributed to inefficient mass transport from the solution to the surface, whereas the immunoreaction itself is a rapid process. The antibodies and reagents used in ELISA are also expensive.
To overcome these drawbacks, industry is miniaturizing and automating ELISA by using 384- or even 1536-well plates and robots to carry out the liquid-handling work. However, the robotic machine is very expensive and not suitable for point-of-use in small diagnostic and testing laboratories. A potential approach is to use microfabricated microfluidic ELISA devices with automatic and reliable (precise) liquid handling functions. Because of their microscale dimensions, the devices can enhance the reaction efficiency, simplify procedures, reduce assay time and sample or reagent consumption, and provide highly portable systems.